Aptamers and antiaptamers

ABSTRACT

The present invention relates to: An aptamer comprising a circular oligonucleotide defining one to four target binding regions; An aptamer comprising an oligonucleotide defining two, three or four thrombin binding quadruplex regions separated by at least partially duplex regions, wherein the quadruplex regions comprise a GGTMGGXGGTTGG sequence wherein M represents A or T and X represents a sequence of two to five nucleotides and/or nucleotide analogues; An aptamer represented by formula (I): 5′D 1 , wQxD 1 D 2 yQzD 2 ,3′—the variables are as defined in the specification; and Aptamers selected from specific sequences.

FIELD OF THE INVENTION

The present invention relates to aptamers, and in particular to aptamershaving circular conformations and thrombin inhibitory activity. Theinvention also relates to compositions comprising such aptamers andmethods of treatment and uses involving the aptamers, as well as toantidotes of aptamer activity.

BACKGROUND OF THE INVENTION

The processes of blood clotting, tissue repair and clot dissolution arereferred to generally as haemostasis, which requires the coordinatedaction of platelets, clotting factors, endothelial cells and smoothmuscle cells within blood vessels (Wu, 1984). Thrombin is an essentialcomponent of the haemostatic processes and is responsible for activationof platelets to adhere to exposed subendothelial structures, conversionof soluble fibrinogen into insoluble fibrin and activation of factorXIIIa, which in turn causes crosslinking of fibrin molecules to form ahard clot.

Apart from its haemostatic functions, thrombin is recognised as having anumber of other activities, for example as a mitogen (Carney et al,1985). It is also thought to exert a chemotactic effect on monocytes(Bar Shavit and Wilner, 1986). In light of these functions thrombin hasbeen implicated as a pro-metastatic agent (Nierodzik et al, 1992) aswell as a factor involved in neurodegenerative disease (Tapparelli etal, 1993). Therefore, apart from the obvious roles of thrombininhibitors in prevention or reduction of thrombosis and blood or bloodproduct coagulation, thrombin inhibitors have the potential to be usedin the treatment of a wide range of disorders including inflammation,cancer and neural disease.

Present anticoagulant and antithrombotic therapies rely upon the use ofheparin and coumarin derivatives that indirectly and incompletelyinhibit the coagulation system. The coumarins are the only class ofcurrently available thrombin inhibitors to possess significant oralactivity, which makes them acceptable to patients and useful in longterm treatments. However, as a result of their mode of action whichinvolves inhibition of hepatic synthesis of vitamin K-dependentcoagulation proteins (Tapparelli et al, 1993), the coumarins areassociated with a number of disadvantages. In particular the coumarinsexhibit pharmacological interactions with food and other drugs, requireseveral days for a full thrombin inhibitory effect to manifest andseveral days for resynthesis of coagulation factors to normalise oncessation of treatment. Coumarin therapy is also characterised byvariability between patients, which necessitates close monitoring.

The most important drugs presently used in the prevention and treatmentof thromboembolic diseases are the heparins, which are administered insurgery and to patients suffering from stroke, acute myocardialinfarction, respiratory failure and during immobilisation of patientswhen extracorporeal circulation or renal dialysis is required (Stubbsand Bode, 1995). Unlike the coumarins, which take days to manifest theireffects, heparin compounds have an immediate effect on bloodcoagulation. However, they are also associated with a wide range ofbiological effects due to their binding of a variety of cells includingplatelets, endothelial cells, red blood corpuscles and lymphocytes(Stubbs and Bode, 1995) as well as an interaction with more than fiftyenzymes (Jaques, 1980). Heparin administration can be associated withside effects including heparin-associated thrombocytopenia andosteoporosis. Although there have been advances with fractionated, moreorally bioavailable heparins, conventional unfractionated heparins arecharacterised by low oral bioavailability which means they must beparenterally administered, such that they are restricted to short termusage. A major, further limitation relating to the heparins is theirineffectiveness in treatment of arterial thrombosis (Topoi et al, 1989).

Although a number of anticoagulant agents have been trialled intreatment of thromboembolic diseases, none so far has supplantedheparin. There is therefore a pressing need to develop anticoagulantagents, that preferably are effective in the treatment of arterialthrombosis, are orally administrable and exhibit long lasting activityin vivo, with minimal side-effects.

Some consideration has been given to the development of nucleic acidaptamers as antithrombotic agents. Aptamers are nucleic acids capable ofthree dimensional recognition that bind specific proteins or othermolecules. Many known thrombin binding aptamers are composed ofoligodeoxynucleotides containing the consensus sequenced(GGTTGGXGGTTGG), (<400>1), where G and T nucleotides are invariant andX is any two to five nucleotides. The 15-mer d(GGTTGGTGTGGTTGG),(<400>2), also known as GS-522 has been the subject of a number ofstructural and functional studies. These known thrombin-binding aptamersare characterised by a central core of two guanine quartets (Guschlbaueret al, 1990) formed from eight conserved guanine residues. These twoG-quartets are linked by two TT loops at one end and a TGT loop at theother end of a quadruplex, as shown in FIG. 1A. Thrombin bindingaptamers of this type have been identified as binding to thrombinexosite II (Padmanabhan et al, 1993). Although GS-522 and molecules likeit have been shown to be effective in inhibiting clot- and matrix-boundthrombin (Li et al, 1994), the antithrombotic aptamers known to datehave been characterised by low oral bioavailability and short in vivohalf life. In an attempt to overcome problems with known thrombinbinding aptamers such as those disclosed in WO 92/14842 a number ofapproaches have been attempted. For example, U.S. Pat. No. 5,399,676proposes DNA-binding oligonucleotides stabilised against exonucleasedegradation by virtue of combining tandem sequences of inverted polarityvia a linker molecule. U.S. Pat. No. 5,668,265 discloses abi-directional nucleic acid ligand that may be used as a diagnostic ortherapeutic and which combines at least two oligonucleotides of oppositesequence polarity via a linker molecule. The publication of Macaya et al(1995) described quadruplex-duplex aptamers stabilized by eitherdisulfide or triethylene glycol (TEG) linkages between the terminalnucleotides. Although molecules of these types demonstrate improvedstability to exonuclease degradation, they are limited in theirtherapeutic and diagnostic utility.

It is with the difficulties associated with prior art antithromboticagents in mind that the compounds according to the present inventionhave been conceived. By virtue of their molecular recognitionproperties, these compounds may also be employed in diagnosticapplications.

SUMMARY OF THE INVENTION

The present invention provides an aptamer comprising a circularoligonucleotide defining one to four target binding regions.

In a preferred form of the invention, the aptamer defines two, three orfour target binding regions.

Preferably, the aptamer defines one or more protein, cellular, cellcomponent or material binding regions.

A preferred cellular binding region is an L-selectin binding domain.

A preferred protein binding region is a thrombin binding region.Accordingly, in one embodiment of the present invention there isprovided an aptamer comprising a circular oligonucleotide defining oneto four thrombin binding regions.

Preferably, the aptamer defines two, three or four thrombin bindingregions wherein said regions are separated by at least partially duplexregions. Preferably, the thrombin binding regions are quadruplexstructures.

According to another embodiment of the invention there is provided anaptamer defining two, three or four thrombin binding quadruplex regionsseparated by at least partially duplex regions, wherein the quadruplexregions comprise a GGTMGGXGGTTGG, sequence (<400>3) wherein M representsA or T and X represents a sequence of two to five nucleotides and/ornucleotide analogues.

Preferably, the aptamer is ligated at its termini to form a circularoligonucleotide. Preferably, the termini have been enzymaticallyligated, or alternatively chemically ligated.

Preferably, X represents a sequence selected from TGT, GCA and TGA.

According to another embodiment of the invention there is provided anaptamer represented by formula I:5′ D₁′wQxD₁D₂yQzD₂′ 3′  Formula Iwherein

-   -   Q represents a sequence GGTMGGXGGTTGG where M represents A or T        and X represents a sequence of two to five nucleotides and/or        nucleotide analogues;    -   w, x, y and z are the same or different and represent a sequence        of zero to ten nucleotides and/or nucleotide analogues;    -   D₁ and D₂ are the same or different and each represent a        sequence of zero to twenty-five nucleotides and/or nucleotide        analogues, with the proviso that D₁ and D₂ together comprise at        least two nucleotides or nucleotide analogues;    -   D₁′ and D₂′ are the same or different and each represent a        sequence of zero to fifty nucleotides and/or nucleotide        analogues, wherein at least two consecutive nucleotides or        nucleotide analogues of D₁′ and/or D₂′ are complimentary to at        least two consecutive nucleotides or nucleotide analogues of D₁        and/or D₂, so as to allow duplex formation between complimentary        nucleotides or nucleotide analogues.

Preferably, the 5′ terminus is phosphorylated.

Preferably, w, x, y and z are the same or different and each representzero, one or two nucleotides and/or nucleotide analogues.

Preferably, D₁ and D₂ in total represent two to twenty nucleotidesand/or nucleotide analogues. Particularly preferably, D₁ and D₂ in totalrepresent four to twelve nucleotides and/or nucleotide analogues.

Preferably, D₁′ and D₂′ in total represent two to twenty nucleotidesand/or nucleotide analogues. Particularly preferably, D₁′ and D₂′ intotal represent four to twelve nucleotides and/or nucleotide analogues.

Preferably, the aptamer is ligated at its termini to form a circularoligonucleotide. Preferably, the termini have been enzymatically ligatedor chemically ligated.

In a preferred embodiment of the invention the aptamer consists ofnucleotides. Preferably, the aptamer consists of RNA and more preferablythe aptamer consists of DNA.

Preferably, X represents a sequence selected from TGT, GCA and TGA.

Preferably, D₁ and D₁′ are selected from the following respective pairs:

-   -   CAG and CTG;    -   CAGC and GCTG;    -   CATGC and GCATG;    -   CATCGC and GCGATG.

Preferably, D₂ and D₂′ are selected from the following respective pairs:

-   -   CAC and GTG;    -   GCAC and GTGC;    -   GCTAC and GTAGC;    -   GACTAC and GTAGTC.

According to another embodiment of the invention there are providedaptamers selected from those comprising the following sequences: DH6-15′ p CTG GGT TGG TGA GGT TGG TCA GCA CGG TTG GTG AGG TTG GTG TG 3′(<400>4) DH8-1 5′ p GCT GTG GTT GGT GAG GTT GGC AGC GCA CTG GTT GGT GAGGTT GGG TGC 3′ (<400>5) DH10-1 5′ p GCA TGT GGT TGG TGA GGT TGG CAT GCGCTA CTG GTT GGT GAG GTT GGG TAG C 3′ (<400>6) DH12-1 5′ p GCG ATG TGGTTG GTG AGG TTG GCA TCG CGA CTA CTG GTT GGT GAG GTT GGG TAG TC 3′(<400>7) TS1-1 5′ p GCT GTG GTT GGT GAG GTT GGC AGC AGC CAA GGT AAC CAGTAC AAG GTG CTA AAC GTA ATG GCT TCG GCT 3′ (<400>8) TT4-1 5′ GAG TCC GTGGTA GGG CAG GTT GGG GTG ACT CGC TGT GGT TGG TGA GGT TGG CAG C 3′(<400>9) TT4-2 5′ GAG TCC GTG GTA GGG CAG GTT GGG GTG ACT CGC TGT GGTTGG TGA GGT TGG ACA GC 3′ (<400>10) TT4-3 5′ GAG TCC GTG GTA GGG CAG GTTGGG GTG ACT CGC TGC GGT TGG TGA GGT TGG GCA GC 3′ (<400>11) DH8-Br1 5′GCT GTG GTT GGB GAG GBB GGC AGC GCA CBG GBB GGB GAG GBB GGG BGC 3′(<400>12)where B=5-bromo-2′-deoxyuridine, 5-iodo-2′-deoxyuridine or otherphotoactive nucleotide analogue.

According to another embodiment of the invention there is provided anantidote aptamer comprising at least ten nucleotides and/or nucleotideanalogues complimentary to a sequence of at least ten nucleotides and/ornucleotide analogues from an aptamer as referred to above.

In one embodiment, the antidote aptamer comprises the followingsequence: ADH8-1 5′ pGCA CCC AAC CTC ACC AAC CAG TGC GCT GCC AAC CTC ACCAAG CAC AGC 3′ (<400>19)

In another embodiment there is provided an antisense oligonucleotide ofan aptamer according to the invention.

In another embodiment there is provided a method of treatment ofthrombosis in a patient requiring such treatment which comprisesadministering to said patient an effective amount of an aptameraccording to the invention.

In another embodiment there is provided a method of preventing orreducing coagulation of blood or blood derived products which comprisescontacting the blood or blood derived product with an effective amountof an aptamer according to the invention.

In another embodiment there is provided use of a compound according tothe invention in preparation of a medicament for the treatment ofthrombosis.

In a further embodiment, there is provided a method for capturingleukocytes from a physiological fluid comprising contacting thephysiological fluid with an effective amount of an aptamer of theinvention.

The invention also provides a composition comprising an aptamer of theinvention together with one or more pharmaceutically acceptable carriersor excipients.

DETAILED DESCRIPTION OF THE FIGURES

The present invention will be described further and by way of exampleonly with reference to the following figures:

FIG. 1 Thrombin aptamer and antidote structures

-   -   (A) Canonical thrombin aptamer    -   (B) Schematic of divalent aptamer with G-quadruplex heads    -   (C) Schematic of divalent antidote aptamer

FIG. 2: Thrombin inhibition of aptamers in selection buffer

Comparative activities of TC, DH and TS aptamer families incubated at37° C. for 1 min in selection buffer. Clotting times represent theaverage of at least three measurements.

Final concentrations of aptamer, thrombin and fibrinogen were 100 nM,˜50 nM and 2 mg/mL, respectively.

FIG. 3: Functional stability of aptamers

(A) Linear and (B) Circular aptamers incubated in 100 μL serum at 37° C.for 1 min and at 1, 6, 12, and 24 h. Clotting was initiated by theaddition of thrombin and fibrinogen in selection buffer. Finalconcentrations: 50 nM DNA, ≈50 nM thrombin and 1.5 mg/mL fibrinogen.

FIG. 4: Physical degradation of aptamers

Incubation of (A) cDH8-1; (B) cTS1-1; (C) cDH12-1; and (D) unligatedpDH12-1 in serum at 37° C. Lanes 1-5 indicate times samples. Circular DHaptamers (A, C) were sampled at 1 min, 1, 6, 12 and 24 h. cTS1-1 (C)samples were collected at 1 min, 1, 2, 3 and 6 h. Unligated pDH12-1 (D)at 1, 15, 30, 60 and 120 min. cDH samples were run on non-denaturingPAGE; cTS1 on denaturing (urea) PAGE. Gels A, B and C were stained withSYBR II for 30 minutes before being visualised under fluorescence. Gel Dwas stained with ethidium bromide for UV luminescence.

FIG. 5: Antidote activity against thrombin aptamers

Fold-anticoagulant activity (ε) for GS-522, pDH8-1 and cDH8-1 (dataavailable for buffer and serum only). Dark-shaded bars indicate ε valuesin the absence of antidote, light-shaded bars in the presence of ADH8-1antidote and hatched bars in the presence of cADH8-1 antidote.

-   -   (A) Buffer    -   (B) Serum    -   (C) Plasma

SEQUENCE LISTINGS

The sequence listings according to the present application include thoseas follows: DH6-1 5′ p CTG GGT TGG TGA GGT TGG TCA GCA CGG TTG GTG AGGTTG GTG TG 3′ DH8-1 5′ p GCT GTG GTT GGT GAG GTT GGC AGC GCA CTG GTT GGTGAG GTT GGG TGC 3′ DH10-1 5′ p GCA TGT GGT TGG TGA GGT TGG CAT GCG CTACTG GTT GGT GAG GTT GGG TAG C 3′ DH12-1 5′ p GCG ATG TGG TTG GTG AGG TTGGCA TCG CGA CTA CTG GTT GGT GAG GTT GGG TAG TC 3′ TS1-1 5′ p GCT GTG GTTGGT GAG GTT GGC AGC AGC CAA GGT AAC CAG TAC AAG GTG CTA AAC GTA ATG GCTTCG GCT 3′ TT4-1 5′ pGAG TCC GTG GTA GGG CAG GTT GGG GTG ACT CGC TGT GGTTGG TGA GGT TGG CAG C 3′ TT4-2 5′ pGAG TCC GTG GTA GGG CAG GTT GGG GTGACT CGC TGT GGT TGG TGA GGT TGG ACA GC 3′ TT4-3 5′ pGAG TCC GTG GTA GGGCAG GTT GGG GTG ACT CGC TGC GGT TGG TGA GGT TGG GCA GC 3′ DH8-Br1 5′pGCT GTG GTT GGB GAG GBB GGC AGC GCA CBG GBB GGB GAG GBB GGG BGC 3′

where B=5-bromo-2′-deoxyuridine, 5-iodo-2′-deoxyuridine or otherphotoactive nucleotide analogue. ADH8-1 5′ pGCA CCC AAC CTC ACC AAC CAGTGC GCT GCC AAC CTC ACC AAG CAC AGC 3′

DETAILED DESCRIPTION OF INVENTION

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.The subject specification contains nucleotide sequence informationprepared using the programme PatentIn Version 3.0, presented hereinafter the references. Each nucleotide sequence is identified in thesequence listing by the numeric indicator <210> followed by the sequenceidentifier (e.g. <201>1, <210>2, etc). The length, type of sequence (egDNA) and source for each nucleotide sequence are indicated byinformation provided in the numeric indicator fields <211>, <212> and<213>, respectively. Nucleotide sequences referred to in thespecification are defined by the information provided in numericindicator field <400> followed by the sequence identifier (e.g. <400>1,<400>2, etc).

The reference to any prior art in this specification is not, and shouldnot be taken as, an acknowledgment or any form of suggestion that thatprior art forms part of the common general knowledge in Australia.

As referred to herein, “a target binding region” is a region within anaptamer that binds to a desired target (eg cell, protein or othermolecule) and thus includes a molecular recognition region within theaptamer which can bind to a target. The terms “region” and “domain” asused herein may be used interchangeably.

In a preferred aspect the present invention relates to an aptamercomprising a circular oligonucleotide defining one to four thrombinbinding quadruplex regions. The aptamers of the present inventiontherefore include oligonucleotides that specifically bind equivalentlyor non-equivalently to molecules such as thrombin and may optionallyinclude sequence motifs that may specifically bind other elements suchas cells, cellular components or other materials such as biomolecules,chromatography columns or beads or the like. The term “oligonucleotide”is intended to encompass nucleic acids including not only those withconventional bases, sugar residues and internucleotide linkages, butalso those that may contain modifications of any or all of thesecomponents. As referred to herein, oligonucleotides therefore includeRNA or DNA sequences of two or more nucleotides in length, (unless thecontext requires otherwise) and may specifically include short sequencessuch as dimers or trimers which may be intermediates in the productionof aptamers according to the invention. Oligonucleotides as mentionedherein encompass those in single chain or duplex form and alsospecifically include those having quadruplex regions, for example of thetype characterised by linked guanine quartets such as exemplified inFIG. 1A. The oligonucleotides forming the aptamers of the presentinvention may constitute DNA (polydeoxyribonucleotides containing2′-deoxy-D-ribose or modified forms thereof), RNA (polyribonucleotidescontaining D-ribose or modified forms thereof) or any other type ofpolynucleotide which is an N-glycoside or C-glycoside of a purine orpyrimidine base, or modified purine or pyrimidine base.

The oligonucleotides according to the present invention may be formed ofconventional phosphodiester-linked nucleotides and synthesised usingstandard solid phase (or solution phase) oligonucleotide synthesistechniques or enzymatic synthesis techniques (with or without primer),which are well known to those skilled in the art. It is also possible,however, for the oligonucleotides of the invention to include one ormore “substitute” linkages as would be well understood in the art.Substitute linkages of this type may for example includephosphorothioate, phosphorodithioate or phosphoramidate type linkages orother modified linkages that would be well understood by persons skilledin the art.

The term “nucleoside” or “nucleotide” encompasses ribonucleosides orribonucleotides, deoxyribonucleosides or deoxyribonucleotides, or othernucleosides which are N-glycosides or C-glycosides of a purine orpyrimidine base, or modified purine or pyrimidine base. Thus, thestereochemistry of the sugar carbons may be other than that of D-ribosein one or more residues. Analogues where the ribose or deoxyribosemoiety is replaced by an alternative structure such as for example a6-membered morpholino ring as described in U.S. Pat. No. 5,034,506 orwhere an acyclic structure serves as a scaffold that positions the baseanalogues are also encompassed. Elements ordinarily found inoligonucleotides such as the furanose ring or the phosphodiester linkagemay be replaced with any suitable functionally equivalent element andmodifications in the sugar moiety, for example wherein one or more ofthe hydroxyl groups are replaced with halogen, or aliphatic groups orare functionalised as ethers, amines and the like, are also included.

The nucleosides and nucleotides of the oligonucleotides according to theinvention may contain not only the natively found purine and pyrmidinebases A, T, C, G and U, but also analogues thereof, which will generallybe referred to as “nucleotide analogues”. Nucleotide analogues may forexample include alkylated purines or pyrimidines, acylated purines orpyrimidines or other heterocycles. The nucleotide analogues encompassedby the present invention are those generally known in the art, many ofwhich are used as chemotherapeutic agents, and examples of which include7-deazadenine, 7-deazaguanine, pseudoisocytosine, N⁴,N⁴-ethanocytosine,8-hydroxy-N⁶-methyladenine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N⁶-isopentenyl-adenine, 1-methyladenine,1-methylpseudouracil, 1-methylguanine, 1-methylinosine,2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine,5-methylcytosine, N⁶-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxy aminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N⁶-isopentenyladenine, uracil-5-oxyacetic acid methylester, pseudouracil, 2-thiocytosine, 5-methyl-2-thiouracil,2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acidmethylester, uracil-5-oxyacetic acid, queosine, 2-thiocytosine,5-propyluracil, 5-propylcytosine, 5-ethyluracil, 5-ethylcytosine,5-butyluracil, 5-butylcytosine, 5-pentyluracil, 5-pentylcytosine, and2,6-diaminopurine. In certain circumstances there may be a call forphotoactive analogues which will degrade on exposure to radiation at anappropriate energy. Examples of photoactive analogues include5-bromo-2′-deoxyuridine and 5-iodo-2′-deoxyuridine. In othercircumstances there may be call for fluorescent nucleotide analogues toenable detection by fluorescence microscopy, fluorescence resonanceenergy transfer (FRET) or other fluorescence detection methodologiesknown to those skilled in the art. In yet other circumstances, there maybe call for electrochemically-labelled nucleotide derivatives to enabledetection by electrochemical methods. Examples ofelectrochemically-labelled nucleotides include ferrocenyl- and metalcomplex derivatives of any nucleotide moiety including 2′-deoxyuridine.The sugar residues of the oligonucleotides of the invention may be otherthan conventional ribose and deoxyribose residues and may for examplecontain analogous forms of ribose or deoxyribose sugars as are wellunderstood in the art. Particular possibilities include sugarssubstituted at the 2′-position of the furanose residue.

As explained above preferred aptamers according to the present inventiondefine one to four thrombin binding quadruplex regions. In a preferredembodiment of the invention the aptamers define two, three or fourthrombin binding quadrupled regions, in which case the quadrupledregions are separated by at least partially duplex regions. That is,within the circular oligonucleotide, regions of complementarity thatdemonstrate base pairing are located between each of the thrombinbinding quadruplex regions. Preferably, the aptamers of the inventioncomprise two or three, most preferably two thrombin binding quadruplexregions. In the situation where the aptamer comprises only a singlethrombin binding quadruplex region it is preferred that the aptamerincludes one or more binding domains that bind cells or cell componentsor other materials.

By the term “thrombin binding quadruplex region” it is intended toencompass a nucleotide sequence having a core of two guanine quartetswhich exhibits specific binding to thrombin. Examples of the nucleotidesequences that define thrombin binding quadruplex regions include theconsensus sequence d(GGTMGGXGGTTGG), where M represents A or T and Xrepresents a sequence of any two to five nucleotides or nucleotideanalogues. In preferred embodiments X may represent TGT, GCA or TGA. Amore specific example is the 15-mer d(GGTTGGTGTGGTTGG), also known asGS-522, as shown in FIG. 1A. Another example of a thrombin bindingquadruplex region is the sequence d(GGTAGGGCAGGTTGG) (<400>13) whichbinds at the heparin-binding exosite (exosite 1). Methods of identifyingspecific thrombin binding oligonucleotides are for example providedwithin WO 92/14842, the disclosure of which is included herein in itsentirety by way of reference.

Within the aptamers of the invention wherein there are two, three orfour thrombin binding quadruplex regions or where there is a singlethrombin binding quadruplex region and one or more cellular, cellcomponent or material binding domains, the various bindingregions/domains are preferably separated by at least partially duplexregions. By this it is intended to convey that within the circularaptamer, and between the various binding regions/domains there is asequence of at least two nucleotides complimentary to a sequence of atleast two nucleotides from another section of the aptamer, whichcomplimentary sequences are configured to allow base pairing and therebythe formation of oligonucleotide that is duplex in the complimentarysections. Preferably each chain of the duplex regions includes two tofifty, more preferably two to twenty and particularly preferably four totwelve nucleotides and/or nucleotide analogues.

In another aspect the invention relates to aptamers that may be utilisedto produce circular aptamers according to the invention. In this regardthe invention also includes single stranded oligonucleotides wherein 5′and 3′ termini may be ligated to produce a circular aptamer. Of course,the non-circular aptamers of this type should include all the necessarycomponents of the aptamers of the invention, namely one to four thrombinbinding quadruplex regions and the optional cellular or other materialbinding domains, in addition to nucleotide sequences that will definethe at least partially duplex regions between the thrombin bindingquadruplex regions and cellular, cellular component or other materialbinding domains if present, when the termini are ligated. Preferably thenon-ligated aptamers are phosphorylated at their 5′ end to therebyprovide the functionality required for enzymatic and/or chemicalligation.

Ligation may involve preferably enzymatic or alternatively chemicalclosure of a phosphorylated open chain oligonucleotide in which the endsare held together by base pairing to a complimentary template sequence(Kool, 1996). Template directed approaches such as this are generallyutilised for cyclisation of oligonucleotides greater than thirtynucleotides in length (Dolinnaya et al, 1993; Prakash and Kool, 1992).Exemplary chemical ligation techniques include the use of a condensingagent such as cyanogen bromide or carbodiimide (Dolinnaya et al, 1988;1991; 1993; Kool, 1991; Fedorova et al, 1995). For example, enzymaticligation may be performed using standard conditions for T4 DNA ligase(Dolinnaya et al, 1988) and circularised DNAs may be purified by use ofdenaturing polyacrylamide gel electrophoresis (PAGE).

It is also possible for other approaches to be adopted in synthesis ofcyclic oligonucleotides including solution methods (Rao and Reese, 1989;Capobianco et al, 1990), polymer supported methods (De Napoli et al,1993) and template-directed approaches (Kool, 1991; Rumney and Kool,1992; Dolinnaya et al, 1993). In the past solution phase approaches havebeen utilised to synthesise small and medium sized oligonucleotides (forexample less than 10 nucleotides in length) and solid phase processeshave been utilised to produce medium sized cyclic oligonucleotides (forexample ten to thirty nucleotides in length).

According to the present invention it is preferred for theoligonucleotides of the invention to be prepared utilising aself-templating approach with oligonucleotides that have internal basepairing (Erie et al, 1989; Ashley and Kushlan, 1991). Thisself-templating approach preferably involves the enzymatic and/orchemical ligation of the duplex region of the aptamer which is formedupon folding.

As previously discussed the aptamers according to the present inventionmay include one or more cellular, cell component or other materialbinding domains which may for example offer utility in assisting uptakeacross the gastrointestinal tract or targeting the aptamers to specificcell types and may offer advantages in linkage to materials such asimplantable biomaterials, components of blood or blood product storageor transfer equipment and diagnostic or filtration equipment components.For example, aptamers of the present invention can be targeted to bindto any of the CD (cluster of differentiation) antigens of which thereare 166 presently known, specific examples of which include L-selectin(CD62L); CD41 and CD42 (located on platelets) and CD44 on leukocytes. Inone preferred embodiment of the invention the circular aptamer includesa domain with binding affinity for L-selectin, a surface protein foundon cells in the circulation, particularly leukocytes (Bradley et al,1992). An advantage that may be associated with aptamers having anL-selectin binding domain is that they can be anchored to circulatingcells which may result in the aptamer being retained within the systemiccirculation. A further advantage arises in capture of leukocytes fromphysiological fluids, especially blood. L-selectin DNA aptamers can begenerated by in vitro selection methods as discussed in Hicke et al(1996), the disclosure of which is included herein in its entirety byway of reference. Three L-selectin aptamers produced according to themethods of Hicke et al (1996), namely LD201, LD174 and LD196, weremodified by removal of bases from each end to generate preserved duplexregions and were attached to the 3′-end of the quadruplex-duplexthrombin aptamers to produce the TS1-1 sequence (amongst others) asreferred to above. While the L-selectin aptamers LD201, LD174 and LD196have little sequence homology they bind L-selectin with comparablenanomolar affinities.

An example of another binding motif that may be incorporated within theaptamers of the invention to provide selective binding to cells is themotif for binding to the cell-surface oligosaccharide cellobiose, asdescribed in Yang et al, 1998, the disclosure of which is includedherein in its entirety by way of reference.

In a particularly preferred embodiment oligonucleotides of the formula Iare utilised to form the circular aptamers according to the invention,wherein formula I is as follows:5′ D₁′wQxD₁D₂yQzD₂′ 3′  Formula I

Within formula I the regions defined as “Q” represent thrombin bindingquadruplex regions having nucleotide sequence GGTMGGXGGTTGG, where Mrepresents A or T and X represents a sequence of two to five nucleotidesand/or nucleotide analogues. In this context it is preferred that Xrepresents TGT, GCA or TGA.

Within formula I the variables w, x, y and z may be the same ordifferent and can represent a sequence of zero to ten nucleotides and/ornucleotide analogues. These variables are intended to representadditional or extraneous nucleotides and/or nucleotide analogues notdirectly within the thrombin binding quadruplex regions and notnecessarily internally complementary. The nucleotides represented by w,x, y and z therefore attribute to bulges or bunching within the circularaptamer and may play a role in directing the orientation of the thrombinbinding quadruplex regions. It is preferred for w, x, y and z torepresent, independently, zero to four nucleotides and/or nucleotideanalogues and it is more particularly preferred for them to representjust zero or one nucleotide or nucleotide analogue. It is most preferredfor one, two, three or four of w, x, y and z to represent a singlenucleotide, which is most preferably T.

The D₁ and D₂ variables may be the same or different and each representa sequence of zero to twenty-five nucleotides and/or nucleotideanalogues, with the proviso that D₁ and D₂ together comprise at leasttwo nucleotides or nucleotide analogues. It is preferred for D₁ and D₂together to represent two to twenty nucleotides and/or nucleotideanalogues, more preferably four to twelve nucleotides and/or nucleotideanalogues.

-   -   The variables D₁′ and D₂′ may be the same or different and each        represent a sequence of zero to fifty nucleotides and/or        nucleotide analogues. However, at least two consecutive        nucleotides or nucleotide analogues of D₁′ and/or D₂′ are        complementary to at least two consecutive nucleotides and        nucleotide analogues of D₁ and/or D₂, so as to allow duplex        formation between complementary nucleotides or nucleotide        analogues. Although it is preferred for the aptamers of the        invention to be somewhat symmetrical in the sense that D₁, D₂,        D₁′ and D₂′ are of the same or at least similar nucleotide        length, this is by no means essential. For example, it is        possible for D₁′ to be two nucleotides in length while D₂′ is        four nucleotides in length and that these six nucleotides are        complementary to six nucleotides defined by D₁ and D₂ in        combination.

As it is intended for D₁′ and D₂′ or at least elements of them to becomplementary with D₁/D₂ or at least elements of the combination, thesense of these elements needs to be reversed to allow complementarity byfolding. Specific examples of respective pairs of D₁ and D₁′ include CAGand CTG; CAGC and GCTG; CATGC and GCATG; CATCGC and GCGATG and specificexamples of D₂ and D₂′ include CAC and GTG; GCAC and GTGC; GCTAC andGTAGC; GACTAC and GTAGTC. A diagrammatic representation of an aptamer ofthe present invention, having two thrombin binding quadruplex regions(T) is shown in FIG. 1B.

Another aspect of the invention relates to antidote (or antisense)oligomers of aptamers of the invention. These may also be referred toherein as “antiaptamers”. Antidote oligomers (or antiaptamers) cancounteract the effect of the corresponding aptamer and thus may beuseful in circumstances where the effect of the aptamer is greater thandesired, for example by using too much aptamer. The antiaptamers arepreferably at least 10 nucleotides and/or nucleotide analogues in lengthand are complementary to a sequence of at least 10 nucleotides and/ornucleotide analogues of an aptamer of the invention.

One embodiment of this aspect relates to antidotes of the thrombinbinding aptamers which comprise aptamers of at least ten nucleotidesand/or nucleotide analogues in length which are complementary to asequence of at least ten nucleotides and/or nucleotide analogues fromwithin a thrombin binding aptamer of the invention. Preferably, theregion of complementarity of the antisense sequence encompasses at leasta portion of one or more of the thrombin binding quadruplex regions.More preferably, the antidote aptamers are complementary to at least aportion of each of the thrombin binding quadruplex regions andparticularly preferably the antidote aptamers constitute an antisenseoligonucleotide to the entire sequence of the thrombin binding aptamerof the invention. A diagrammatic representation of an antidote aptameris shown in FIG. 1C.

The invention thus also provides a method for counteracting the effectof an aptamer of the invention comprising contacting the aptamer with acounteracting effective amount of its antiaptamer.

As used herein, “counteracting” refers to the inhibition, halting orpartial or full reversal of the effect of the aptamer.

Aptamers according to the present invention and their antisenseantidotes may be formulated into standard pharmaceutical dosage forms bycombination with one or more pharmaceutically acceptable carriers and/orexcipients. Examples of pharmaceutically acceptable carriers andexcipients are provided within Remington's Pharmaceutical Sciences, 17thEdition, Mack Publishing Co, Easton, Pa., USA, the disclosure of whichis included herein in its entirety, by way of reference. Although it ispreferred for the aptamers of the present invention to be formulatedinto oral dosage forms, it is additionally possible for formulation intoforms suitable for intravenous, intramuscular, subcutaneous, buccal,intraperitoneal, rectal, vaginal, nasal and ocular delivery, forexample.

As used herein “pharmaceutically acceptable carrier and/or excipient”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredient, use thereofin the therapeutic compositions is contemplated. Supplementary activeingredients may also be incorporated within the compositions of theinvention. Dosage forms according to the present invention which may forexample be formulated as tablets, troches, pills, capsules, injectables,salves, ointments, drops, sprays, powders and the like will preferablybe formulated into unit dosage forms, which may for example containbetween about 0.1 μg and 2,000 mg of active compound. As will be wellrecognised by a skilled medical practitioner or pharmacist the-effective dosage of the active ingredients according to the presentinvention will be dependent upon the nature of the disorder beingtreated and the height, age, weight, sex and general fitness of thepatient concerned.

The aptamers according to the present invention are particularly suitedfor treatment and/or prevention of thrombosis, stroke, myocardialinfarction and respiratory failure. The aptamers according to theinvention may also be utilised in prevention of clotting as a result oftrauma, and may be used in surgery, in the treatment and/or preventionof inflammatory disorders, cancer metastasis, neural disease and bloodcoagulation. In the case where rapid reversal of action of the aptamersaccording to the invention is required, for example in the situation ofan overdose, it is possible to administer the antidote aptamers in anamount sufficient to bind the thrombin binding aptamers andcompetitively inhibit their activity.

The aptamers according to the present invention may also be utilised inthe prevention of blood or blood product coagulation by theirincorporation within or addition to blood sample tubes and bags or othermaterials that blood or blood products such as serum or plasma may comeinto contact with. In preferred embodiments of the invention aptamersincluding material binding domains may be incorporated into materialssuch as implantable biomaterials including stents, prostheses and thelike to prevent localised blood clotting. The aptamers may also beutilised in conjunction with tissue and/or organ transplants and/orxenotransplants, particularly in relation to vascular grafts. Theaptamers according to the invention may also be utilized in the captureof leukocytes from physiological fluids, especially blood, as part of amedical or genetic diagnostic procedure.

The invention will now be further described with reference to thefollowing non-limiting examples.

EXAMPLES Example 1 Preparation, Circularisation and Isolation ofAptamers

Materials

General Reagents

N-2-hydroxy-ethylpiperazine-N′-2-ethane (HEPES, Sigma Chemical Co.),spermidine (Sigma Chemical Co), tris acetate (BDH),2(N-morpholino)ethanesulfonic acid (MES; Sigma Chemical Co.),3,3′-deithyl-9-methyl-4,5,4′5′-dibenzothiacarbocyanine (STAINS-ALL;Sigma Chemical Co.), TE-saturated phenol/chloroform pH 8 (Progen),Dithiothreitol (DTT; Progen), ammonium persulphate (APS; Sigma ChemicalCo.), boric acid (BDH), potassium chloride (BDH), Tris (Ajax Chemicals),magnesium chloride (BDH), calcium chloride (BDH), glycerol (AjaxChemicals), β-mercaptoethanol (Ajax Chemicals) and adenosine 5′triphosphate (ATP; Sigma Chemical Co.). Bio-Spin P6 and P30 columns,N,N,N′N′-tetramethylethylenediamine (TEMED), 40% bisacrylamide solutionand ethidium bromide were purchased from Bio-Rad Laboratories. SYBRGreen II RNA stain was purchased from Molecular Probes. All reagentswere of analytical grade and all solutions were prepared with Milli-Qdeionised water.

Enzymes and Associated Materials

Calf intestine alkaline phosphatase (MBI Fermentas), human α-thrombin3700 U/mg (Sigma Chemical Co.), bovine α-thrombin 5000 U (ArmourPharmaceutical Co.), T4 DNA Ligase (MBI Fermentas) and cyanogen bromide(Sigma Chemical Co.) were used as received.

DNA Oligonucleotide Sequences

Sequences for the quadruplex-duplex thrombin aptamers were developedfrom oligonucleotides studied by Macaya et al. (1995). MFOLD (Zucker,1994) was used to determine sequence secondary structure. Set A containsthe classic aptamer GS-522 and thrombin circle (TC) family; Set Bcontains the double header (DH) family; and Set C contains thethrombin-selectin (TS) familyoligonucleotides. A modified version of DH8was synthesised with 5-bromo-2′-deoxyuridine phosphoramidite (GlenResearch) substituted for six T residues (B=5-bromo dU). DH8-Br was usedfor photocrosslinking. All oligonucleotides except GS-522 werephosphorylated at the 5′-end using phospholink reagent (Perkin Elmer).Oligonucleotides were deprotected and gel purified before use. SET AGS-522 15-mer 5′ GGT TGG TGT GGT TGG 3′ TC1 5′-P 27mer 5′ p ACT GGT TGGTGA GGT TGG GTG CGA AGC 3′ (<400>14) TC1-T 5′-P 26mer 5′ p ACG GTT GGTGAG GTT GGG TGC GAA GC 3′ (<400>15) TC3 5′-P 28mer 5′ p ACT GGT TGG TGAGGT TGG GTG CGA AAG C 3′ (<400>16) SET B DH6-1 5′-P 44mer 5′ p CTG GGTTGG TGA GGT TGG TCA GCA CGG TTG GTG AGG TTG GTG TG 3′ DH8-1 5′-P 48mer5′ p GCT GTG GTT GGT GAG GTT GGC AGC GCA CTG GTT GGT GAG GTT GGG TGC 3′DH10-1 5′-P 52mer 5′ p GCA TGT GGT TGG TGA GGT TGG CAT GCG CTA CTG GTTGGT GAG GTT GGG TAG C 3′ DH12-1 5′-P 56mer 5′ p GCG ATG TGG TTG GTG AGGTTG GCA TCG CGA CTA CTG GTT GGT GAG GTT GGG TAG TC 3′ SET C TS1-1 5′-P69-mer 5′ p GCT GTG GTT GGT GAG GTT GGC AGC AGC CAA GGT AAC CAG TAC AAGGTG CTA AAC GTA ATG GCT TCG GCT 3′ TS2-1 5′-P 69mer 5′ p GCT GTG GTT GGTGAG GTT GGC AGC AGC TGG CGG TAC GGG CCG TGC ACC CAC TTA CCT GGG AAG TGAGCT 3′ (<400>17) TS3-1 5′-P 69mer 5′ p GCT GTG GTT GGT GAG GTT GGC AGCAGC CAT TCA CCA TGG CCC CTT CCT ACG TAT GTT CTG CGG GTG GCT 3′ (<400>18)MODIFIED OLIGONUCLEOTIDE DH8-Br1 5′-P 48mer B = 5-bromo-2′-deoxyuridine5′ p GCT GTG GTT GGB GAG GBB GGC AGC GCA CBG GBB -GGB GAG GBB GGG BGC 3′MethodsSterilisation of Materials

All heat labile solutions were sterilised by filtration through 0.2 μmcellulose acetate disposable filters (Millipore). All other solutionswere sterilised by autoclaving for 30 min (1.0 kg cm⁻², 120° C.).Disposable microfuge tubes and spin columns were all sterilised byautoclaving. Biological waste was autoclaved prior to disposal. Allother waste was disposed of in accordance with the regulationsrecommended by the UNSW Safety Unit.

Cleavage and Deprotection of Synthesised Oligonucleotides

Concentrated ammonium hydroxide solution (5 M) was applied to thesynthesis column using a 1 mL syringe. Columns were inverted and severalaliquots of ammonium hydroxide solution were passed through the columnover a 1 h period at room temperature. The solution was then expressedinto a screw cap tube and placed in a water bath at 55° C. overnight.After incubation, the tubes were dried under vacuum in a Speed-Vac SC110(Savant Instruments Inc.) and redissolved in 100 μL sterile water.Samples were then purified by gel electrophoresis.

Phenol Extraction and Ethanol Precipitation

Protein was removed from aqueous samples by extraction with an equalvolume of buffered phenol (Tris, pH 8.0). The DNA was concentrated byprecipitation with ice-cold ethanol added at 2.5×the volume of aqueoussample after addition of one-tenth sample volume of 3 M sodium acetate.After mixing, samples were left at −20° C. for one hour and immediatelycentrifuged at 10 000×g (4° C.) for 15-20 min. Precipitated DNA waswashed with 1 mL 95% ethanol and centrifuged again at 10 000×g for afurther 2 min. The supernatant was removed and the pellets dried byvacuum centrifugation in a Speed-Vac SC110 vacuum concentrator.

Serum Isolation

Whole blood (20 mL) was clotted at 37° C. for 5 min. Clotting wasinitiated by contact with a glass slide. Serum was collected bycentrifugation at 3000 rpm for 20 minutes in a Clements GS100 swing-outcentrifuge. Serum samples (2 mL) were stored at −70° C. All bloodproducts were handled in accordance with UNSW biological hazardguidelines.

Gel Electrophoresis

Gel Purification

Newly synthesised ssDNA and circular ssDNA were purified by 20%denaturing PAGE. Approximately 50-100 μg of nucleic acid was loaded ontoeach lane of a 10 cm×8 cm×0.15 cm gel in loading buffer not containingtracking dyes. A target product marker was also loaded with buffercontaining tracking dyes in order to facilitate both estimation ofrunning time and identification of correct products. Gels were run at aconstant voltage of 100 V in 1×TBE buffer (pH 8.0) on a Mini-Protean IIgel electrophoresis apparatus (Bio-Rad Laboratories). Gels were stainedfor 15 min in RO water (100 mL) containing ethidium bromide (0.5 μg/mL).Nucleic acids were visualised by UV shadowing and the bands excisedusing sterile implements. Nucleic acids were eluted from crushed gelfragments overnight by diffusion at 37° C. in a solution of 0.3 M NaCl,10 mM Tris-HCl and 1% (v/v) phenol. Targets were collected via ethanolprecipitation and dried in a vacuum concentrator. The dry samples wereredissolved in sterile water and further purified in a Bio-Spin P6column. Final nucleic acid concentrations were determined by UVspectrophotometry.

SDS PAGE

SDS PAGE consisting of a 10% resolving and 4% stacking gel was used todetermine the purity and approximate quantity of protein. A stocksolution containing broad range size markers was diluted 20× in SDSreducing sample buffer (0.5 M Tris-HCl, pH 6.8, 10% glycerol, 10% SDS,0.1% bromophenol blue, β-mercaptoethanol). 10 μL protein samples weremixed in 5 μL sample buffer. All samples were heated to 90-95° C. for 5min before loading onto the gel. Gels were run in SDS/Tris-HCl at 50 mAfor 1 h. Electrophoresed gels were stained with 0.1% coomassie blue forhalf an hour and destained for 1-3 hours in 40% methanol/10% acetic acidand dried overnight.

Agarose Gel

Agarose gels were used to determine the activity of preparative T4 DNAligase after purification. Lambda phage DNA standards (2 μg) and T4 DNAligase treated samples in loading dye were run on 1% agarose gels (0.5 gagarose, 49.5 mL H₂O) in 1× TBE buffer an 100 V for 1 h. Gels werestained with ethidium bromide (0.5 μg/mL) for 30 min and visualised byUV illumination.

UV Spectrophotometry

DNA Quantitation

DNA was quantified by measuring the absorbance of a suitably dilutedsample at 260 nm using a JASCO V-530 UV/VIS Spectrophotometer. Puritywas gauged by the ratio of absorbance between 260 nm and 280 nm. Thefollowing calculation was used to estimate DNA concentration[DNA]=A ₂₆₀×dilution factor×33 μg/mL×sample volumeMelting Profiles

Absorbance versus temperature profiles were measured at a wavelengthcorresponding to the average maximum absorbance achieved at 5 and 95° C.using the JASCO v530 spectrophotometer interfaced to a PC. Meltingprofiles were obtained by increasing the temperature from 5-95° C. at aconstant rate of 0.8° C./min with a programmable,thermoelectrically-controlled cell holder. Melting profiles of samples(OD˜0.5) were performed in 100 mM K₃PO₄ buffer pH 7.5 andcircularisation buffer (1 M MES/0.02 M MgCl₂, pH 7.5). First derivativesof melting curves were used to calculate melting temperature (T_(m)).

Mass Spectroscopy

Oligonucleotide 5′-phosphorylation was determined using MALDI-TOF massspectrometry (The Voyager). Oligonucleotides were first desalted usingAGSOW-X8 NH₄ ⁺ resin (Bio-Rad). Sterile water (0.5-1 μL) containing ofeach oligonucleotide (1-10 pmol) and picolinic acid matrix (0.5-1 μL of400 mM) were mixed and then applied to a metallic target. Negative ionmass spectra were used to detect aptamers. Analysis was performed usingGRAM and MONITOR software.

Circularisation

Enzymatic Ligation: T4 DNA Ligase

Oligonucleotides were heated in selection buffer (100 mM KCl, 1 mMMgCl₂, 20 mM Tris acetate, pH 7.4; Macaya et al, (1995)) to 75-85° C.and slowly cooled to 0-5° C. over 30-60 min. T4 DNA Ligase buffer (1×)and T4 DNA ligase (5 U/μg DNA) were added in the presence or absence ofbovine α-thrombin (20%). The reaction mix was placed at 15-25° C.for >16 h. This was immediately followed by phenol extraction andethanol precipitation. Ligation products were analysed on 20% PAGE.

Larger scale circularisation experiments were conducted in a similarfashion to the above procedure, however, ligations did not containthrombin. Reactions were incubated at room temperature for periods up to4 days. Circular products were subsequently obtained using gelpurification.

Chemical Ligation: Cyanogen Bromide

This procedure was modified from the cyanogen bromide ligation describedby Dolinnaya et al. (1993) and Fedorova et al. (1995). Oligonucleotidein 10× vol buffer (0.25-0.5 M MES-(C₂H₅)₃N, pH 7.5, 0.02 M MgCl₂, withor without 50 mM KCl) was heated to 85° C. for 2 min and slowly cooledto 0-5° C. over 30 min. BrCN in acetonitrile (5 M) was added to thesamples on ice at one-tenth of the volume of the reaction mix. Finalconcentrations of the oligonucleotide and BrCN were 50 μM and 0.5 M,respectively. Reactions were incubated on ice for 5 min. Uponcompletion, the reaction was quenched by addition of 2.5× vol of 100%ice-cold ethanol. Samples were ethanol precipitated and analysed by 20%denaturing (urea) PAGE. This method was used in both small and largescale circularisation procedures.

Exonuclease Treatment of Circular Product

Pellets from circularisation experiments were redissolved in T4 DNApolymerase buffer. T4 DNA polymerase (6 Units/μg DNA) was added to thesamples and reaction tubes were placed at 25° C. for 24 h. Protein wasremoved by phenol extraction followed by ethanol precipitation. Resultswere analysed on 20% denaturing (urea) PAGE.

Example 2 Thrombin Inhibition Assays

All clotting times were estimated using a fibrometer (BehringDiagnostics).

Methods

Selection Buffer

The assay for inhibition of thrombin-catalysed fibrin clot formation inserum free medium was modified from Macaya et al, (1995). Humanfibrinogen in selection buffer (140 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1 mMCaCl₂, 20 mM Tris acetate, pH 7.4, 200 μL) was equilibrated at 37° for 1min in the presence of each oligonucleotide. Reactions were initiated bythe addition of bovine α-thrombin (100 μL in selection bufferpreequilibrated to 37° C. for 5 min). Final concentrations of 2 mg/mLfibrinogen and 100 nM oligonucleotide were reached. Thrombinconcentration varied from 50-100 nM to achieve a baseline (nooligonucleotide present) clotting time of approximately 30-40 s.

Serum

Conditions for serum assays were taken from Macaya et al. (1995).Oligonucleotides were incubated in serum (100 μL) at 37° C. for 1 min.Clotting was initiated by the addition of fibrinogen (200 μL) andthrombin (100 μL) in selection buffer pre-equilibrated to 37° C. Finalconcentrations of 50 nM DNA, 1.5 mg/mL fibrinogen and 50-100 nM thrombinwere achieved with a baseline clotting time of between 30-40 s.

Results

Aptamer families were tested for their ability to inhibit thrombin usingstandard activity assays. Thrombin aptamers inhibit thrombin-activatedclot formation by binding to the fibrinogen recognition site of theenzyme, preventing fibrinogen being cleaved into fibrin. Initially,anti-thrombin activity of aptamers was examined in the absence of bloodproducts. The simplest system involves the isotonic cell- andprotein-free environment of the selection buffer used for the aptamerisolation. Aptamers (100 nM) were incubated in selection buffercontaining a fixed concentration of fibrinogen (2 mg/mL) at 37° C.Clotting is initiated by the addition of thrombin and the time taken forclot formation is conventionally measured using a fibrometer orcoagulator. Thus, activity of the aptamer is defined in terms ofclotting time.

Activity in Selection Buffer

Clotting times for each oligonucleotide family in selection buffer arepresented in Table 1 and FIG. 2. These times were compared to theclassic thrombin aptamer (GS-522) as positive control, and negativecontrols consisted of a telomere construct (H42) and an 18-mer DNAprimer (P3A1). Clotting time in the presence of thrombin and fibrinogenonly (no aptamer) is used as a baseline measurement.

Comparison of Aptamers

The majority of aptamers exhibited inhibition of thrombincatalysed-fibrin clot formation. With the exception of DH6-1, DHaptamers (unligated and circular) showed the greatest thrombininhibition of the three families with clotting times at least three-foldhigher than the classic aptamer GS-522 and up to ten times the baseline.Circular aptamers were generally observed to be somewhat betterinhibitors than unligated species (Table 1; FIG. 2). TABLE 1 ThrombinInhibition by Aptamers Clotting Time [s]^(a) Selection buffer^(b)Serum^(c) Oligo Unligated Circular Unligated Circular GS-522 161(3)  nd70(1) nd DH6-1 54(6)  49(5) nd nd DH8-1 279(16) 449(1) 95(7)  352(35)DH10-1 356(13) 413(5) 82(3) 186(4) DH12-1 374(6)   454(44) 121(4) 231(3) TS1-1 53(3) 236(1) 44(1) 101(2) No Aptamer^(d) 41(2) 38(1)^(a)oligonucleotides incubated 1 min in media (selection buffer orserum) at 37° C. Tabulated values represent the averages of at leastthree measurements; standard errors in parentheses.^(b)selection buffer: 100 nM DNA; 2 mg/ml fibrinogen; 2 × thrombin^(c)serum: 50 nM DNA; 1.5 mg/ml fibrinogen; 1 × thrombin^(d)baseline activitynd: not determinedTC Family

Linear TC aptamers exhibited low activities (70-140 s) when compared toGS-522 (160 s). However, these clotting times are at least double thebaseline time (≈40 s), indicating aptamers have thrombin inhibitoryactivity even though melting profiles suggest that they do not fold. Theobserved clotting times are believed not to be due to non-specificinhibition as TC aptamer activities are significantly more active thanthe negative controls (H42, P3A1). The loss of the T residue (TC1-T) hadno marked effect on inhibition. The longer loop of TC-3 improvedclotting time, doubling the activities of triloop oligonucleotides(TC1-T), but was not greater than the activity of GS-522.

DH Family

In most cases, unligated DH aptamers exhibited relatively highactivities, 2-3 fold higher than GS-522 and at least five times higherthan the baseline clotting time (FIG. 2). DH6-1 was the only unligatedspecies not to exhibit clotting inhibition. Thrombin inhibition alsogenerally increased with increasing duplex length and meltingtemperature such that the following trend was observed:DH6-1<<DH8-1<DH10-1<DH12-1. However, activity and thermal stability (Tm)tended to plateau at longer duplex sizes (DH10-1 and DH12-1; FIG. 2).

Circularised species (except cDH6-1) increased baseline clotting timemore than 10-fold (450 s cf. 40 s) and displayed at least three timesthe activity of the classic aptamer GS-522 (FIG. 2). Given standarderror in the activities of cDH8-1, cDH10-1 and cDH12-1 (Table 1)differences in clotting times are not considered to be significant.Unlike other cDH aptamers, cDH6-1 did not inhibit clotting and exhibitedsimilar activity to the negative controls.

TS Family

Of the linear TS species, only TS2-1 demonstrated significant thrombininhibition (t≈70 s), however this is less than half the activity ofGS-522. Both TS1-1 and TS3-1 exhibited clotting inhibitions similar tothe negative controls. Circularisation increased activity of linearTS1-1 by 200 s. The clotting inhibition of cTS1-1 is two-fold higherthan GS-522 and similar to unligated DH8-1.

Activities in Serum Supplemented Media

To investigate the potential use of aptamers as anticoagulants inmammals, the ability of these oligonucleotides to inhibit thrombin invitro using serum supplemented media was examined. Serum (pre-clottedcell-free fluid) simulates to a certain extent in vivo conditions byproviding molecules and proteins, such as exo- and endo-nucleases,required for a more complete (although more complex) analysis of aptameractivity. Human serum is used in this study rather than 10% fetal calfserum (FCS; Macaya et al., 1995) to provide a better assessment ofaptamer performance in the intended target species.

Clotting assays using serum (Macaya et al., 1995) were performed in asimilar way to the previous assay, however, aptamers were incubated inserum for 1 min at 37° C., before fibrinogen and thrombin addition.Final concentrations of DNA and fibrinogen were also reduced (50 nM DNA;1.5 mg/ml fibrinogen cf. selection buffer) due to limited reagents.However, the effect of serum on aptamers can be generally examined andcompared to selection buffer.

As can be seen in Table 1, circular aptamers are better inhibitors ofthrombin in serum, with at least two-fold higher activities than theirunligated counterparts. This higher activity is not as significant inselection buffer (except cTS1-1 as linear TS1-1 exhibited no activity ineither medium). cDH8-1 has a much higher anti-thrombin activity than theother cDH aptamers (350 s cf.<240 s), which is not observed forunligated DH8-1 in serum. All unligated DH oligonucleotides have similarserum clotting times. As the standard error for cDH8-1 is large, it issuspected that the actual activity may be lower than the tabled value.

Example 3 Serum Stability

Methods

Functional Stability Assay

Oligonucleotides were incubated in human serum (500 μL) at 37° C. and100 μL samples were taken at 1 min and at 1, 6, 12 and 24 h. Sampleswere assays by the addition of fibrinogen (200 μL in selection buffer;37° C.) followed by bovine thrombin (100 μL in selection buffer; 37° C.)to initiate the clotting reaction. Final concentrations of reagentswere: 50 nM oligonucleotide, 1.5 mg/mL fibrinogen and 50-100 nM thrombinto achieve a baseline clotting time of between 30-40 s.

Physical Stability: PAGE

Oligonucleotides (2 μg) were added to serum (100 μL) and incubated at37° C. At different time intervals 20 μL samples were taken and thereaction quenched with 20 μL phenol/chloroform pH 8. An aliquot (2× vol)of Tris-HCl (10 mM, pH 8) was also added before vortexing thoroughly.Samples were centrifuged at 14 000 rpm, 4° C. for 5 min and the aqueouslayer removed. The phenol layer was re-extracted with Tris-HCl. Combinedaqueous layers were ethanol precipitated and run of 20% native ordenaturing PAGE. Gels were stained with SYBR Green II (1:10 000 1× TBE)for 30 min. Gels were then subjected to image analysis.

Image Analysis

PAGE gels from serum stability studies were analysed using Fluoro-SMulti-Imager (Bio-Rad). UV scanning illumination (320 nm) was used withthe lens aperture fully open. Analysis of gel images was performed usingMulti-Analyst/Macintosh software Version 1.0 (Bio-Rad).

Results

To investigate the susceptibility of circularised aptamers tonucleolytic activity, oligonucleotides were incubated in serum andexamined for both functional and physical stability. Functionalstability describes the ability of oligonucleotides to maintain theirinhibition of thrombin-catalysed fibrin clot formation over time.Physical stability refers to actual nuclease degradation of aptamers asdemonstrated by PAGE. Note that serum stability measurements were onlytaken for those unligated oligonucleotides that were circularised inhigh yields and exhibited significant thrombin inhibition in selectionbuffer (ie. DH8-1, DH10-1, DH12-1 and TS1-1).

Functional Stability

Aptamers were incubated in serum at 37° C. and their activity analysedat 1 min and 1, 6, 12 and 24 h. The results for unligated and circularaptamers are shown in FIG. 3. The classic aptamer GS-522 displayed noactivity after one hour in serum, which is consistent with previousreports on its low serum stability (Griffin et al., 1993; Macaya et al.,1995). In FIG. 3A, unligated TS1-1 exhibits very little thrombininhibition, similar to its performance in selection buffer. DH unligatedaptamer activities decline over the first 6 hours to an activityapproximately equal to the initial clotting time of GS-522. Differencesin clotting inhibition of unligated DH oligonucleotides over the 24 hperiod are not thought to be significant. At 12 to 24 h there is littleaptamer activity observed, however, activities do not reach baselinelevels within the 24 h period.

Circular aptamers (except cTS1-1) maintained significant clottinginhibition over 24 h (FIG. 3B). At 24 h, cDH oligonucleotides exhibitedclotting times equal to (cDH10-1, cDH12-1) and greater than (cDH8-1) the1 min clotting time of the classic aptamer GS-522. cDH8-1 demonstratedthe greatest clotting inhibition at each time point, with valuesapproximately twice those of cDH10-1 and cDH12-1. The latter aptamerpair had similar activity values (Table 1) which were about four foldhigher than cTS1-1. However, as this activity of cDH8-1 is significantlygreater than that observed in selection buffer (taking concentrationsinto account), experimental error is suspected (Table 1).

The activity of all the circular aptamers showed a similar pattern offunctional decay, which was different to the functional decay observedfor the unligated species. In FIG. 3B, a rapid decrease in activity inthe first hour is followed by a slow decline to 24 h. Beyond the firsthour, aptamer activity decay corresponds well to first order kineticsand half-lives were determined using first order rate constants (Table2). Unligated and circular DH aptamers tend to have similar functionalhalf-lives.

According to the kinetic data, unligated and circular DH aptamersmaintain their activity in serum up to seventy times longer than GS-522.The half-life of cTS1-1 is somewhat dubious since determination ofhalf-lives requires 2-4 half-lives to be followed to ensure accuracy.The half-life of unligated TS1-1 could not be determined as it showedpoor initial activity. TABLE 2 Kinetics of Functional Decay APTAMER k₁(s⁻¹)^(a) k₂ (s⁻¹)^(b) t_(1/2) (h)^(c) pDH8-1 2.7 × 10⁻⁵ 9.9 × 10⁻⁶ 19.4pDH10-1 2.8 × 10⁵   1.3 × 10⁻⁵ 15.2 pDH12-1 3.2 × 10⁻⁵ 1.1 × 10⁻⁶ 18.3pTS1-1 3.8 × 10⁻⁵ —d —d cDH8-1 9.0 × 10⁵   9.1 × 10⁻⁶ 21.1 cDH10-1 8.6 ×10⁻⁵ 1.4 × 10⁻⁵ 13.7 cDH12-1 9.6 × 10⁻⁵ 1.2 × 10⁻⁵ 15.7 cTS1-1 1.3 ×10⁻⁴ 4.5 × 10⁻⁶ 42.7^(a)Initial rate constant k₁ is determined from the slope of a linearplot of ([A]_(t) − [A]₀ vs. time (two data points).^(b)The rate constant k₂ is determined from the slope of a plot ofIn([A]_(t)/[A]₀) vs (4 data points).^(c)Half-life, t_(1/2) = In2/k₂dnot determinedPhysical (Nuclease) Stability

Physical analysis was performed using PAGE to investigate whether theinitial high activity loss in the first hour of the functional assay wasdue to physical degradation or to another factor such as non-specificprotein binding. Circular aptamers were incubated in serum at 37° C. andsampled at different time points (1 min, 1, 2, 6, 12 and 24 h). PAGEresults for cDH aptamers and cTS1-1 (FIG. 4) suggest physicaldegradation is not responsible for the initial sharp fall in activity(FIG. 3B). Little degradation (unligated product) can be observed in thefirst hour and there is significant circular product at 6 (cTS1-1) and24 h (cDH aptamers), indicating that circular aptamers are extremelystable in comparison to GS-522. The length of the duplex region wasfound to influence nuclease stability as cDH10-1 and cDH12-1 weredegraded more quickly than cDH8-1 (FIG. 4A, C). Unligated DH12-1 wasalso tested for physical stability and found to be more susceptible tonuclease activity, degrading faster than cDH12-1 (FIG. 4D).

Example 4 ADH8-1 Antidote Aptamer Experiment

To investigate the potential of antisense hybridisation as a generalaptamer antidote mechanism, the pADH8-1 reverse complement of DH8-1 wasprepared. This construct contained an internal 8 bp duplex with tworelatively unstructured C-rich heads (as depicted diagrammatically inFIG. 1C), as indicated by an experimental melting temperature of 31° C.

The unligated and circular forms of ADH8-1 displayed almost identicalphysical half-lives of 4 h in serum and 5 h in plasma. These values wereconsistent with a significant protective effect from the internalduplex, but a greater nuclease susceptibility than the DH constructs dueto the absence of tightly folded head motifs.

The effect of aptamer topology on antidote effectiveness was furtherinvestigated by incubating aptamer/antiaptamer mixtures in serum for 10min before fibrometer assay. This is a reasonable upper limit for auseful antidote effect. As shown in FIG. 5, interactions in which atleast one of the partners was unligated displayed a complete antidoteeffect at an antidote:aptamer ratio of 2:1.

It is to be recognised that the present invention has been described byway of example only and that modifications and/or alterations theretowhich would be apparent to persons skilled in the art, based upon thedisclosure herein, are also considered to fall within the spirit andscope of the invention.

REFERENCES

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1. An aptamer comprising a circular oligonucleotide defining one to fourtarget binding regions.
 2. The aptamer of claim 1 which defines two,three or four target binding regions wherein said binding regions areseparated by at least partially duplex regions.
 3. The aptamer of claim1 which defines one or more protein, cellular, cell component ormaterial binding region.
 4. The aptamer of claim 3 wherein the proteinbinding region is a thrombin binding region
 5. The aptamer of claim 3wherein the cellular binding region is an L-selectin binding domain. 6.The aptamer of claim 1 consisting of nucleotides.
 7. The aptamer ofclaim 1 consisting of RNA.
 8. The aptamer of claim 1 consisting of DNA.9. An aptamer comprising an oligonucleotide defining two, three or fourthrombin binding quadruplex regions separated by at least partiallyduplex regions, wherein the quadruplex regions comprise a GGTWGGXGGTTGG(SEQ ID NO:3) sequence wherein M represents A or T and X represents asequence of two to five nucleotides and/or nucleotide analogues.
 10. Theaptamer of claim 9 ligated at its termini to form a circularoligonucleotide.
 11. The aptamer of claim 10 wherein the termini havebeen chemically ligated.
 12. The aptamer of claim 10 wherein the terminihave been enzymatically ligated.
 13. The aptamer of claim 9 consistingof nucleotides.
 14. The aptamer of claim 9 consisting of RNA.
 15. Theaptamer of claim 9 consisting of DNA.
 16. The aptamer of claim 15wherein X represents a sequence selected from TGT, GCA and TGA.
 17. Anaptamer represented by formula I:5N D₁NwQxD₁D₂yQzD₂N 3N   Formula I wherein Q represents a sequenceGGTWGGXGGTTGG (SEQ ID NO:3) where M represents A or T and X represents asequence of two to five nucleotides and/or nucleotide analogues; w, x, yand z are the same or different and represent a sequence of zero to tennucleotides and/or nucleotide analogues; D₁ and D₂ are the same ordifferent and each represent a sequence of zero to twenty-fivenucleotides and/or nucleotide analogues with the proviso that D₁ and D₂together comprise at least two nucleotides or nucleotide analogues; D₁Nand D₂N are the same or different and each represent a sequence of zeroto fifty nucleotides and/or nucleotide analogues, wherein at least twoconsecutive nucleotides or nucleotide analogues of D₁N and/or D₂N arecomplimentary to at least two consecutive nucleotides or nucleotideanalogues of D₁ and/or D₂, so as to allow duplex formation betweencomplimentary nucleotides or nucleotide analogues.
 18. The aptamer ofclaim 17 wherein the 5N terminus is phosphorylated.
 19. The aptamer ofclaim 17 wherein w, x, y and z are the same or different and eachrepresent zero, one or two nucleotides and/or nucleotide analogues. 20.The aptamer of claim 17 wherein D₁ and D₂ in total represent two totwenty nucleotides and/or nucleotide analogues.
 21. The aptamer of claim20 wherein D₁ and D₂ in total represent four to twelve nucleotidesand/or nucleotide analogues.
 22. The aptamer of claim 17 wherein D₁N andD₂N in total represent two to twenty nucleotides and/or nucleotideanalogues.
 23. The aptamer of claim 22 wherein D₁N and D₂N in totalrepresent four to twelve nucleotides and/or nucleotide analogues. 24.The aptamer of claim 17 ligated at its termini to form a circularsequence of nucleotides and/or nucleotide analogues.
 25. The aptamer ofclaim 24 wherein the termini have been chemically ligated.
 26. Theaptamer of claim 24 wherein the termini have been enzymatically ligated.27. The aptamer of claim 17 consisting of nucleotides.
 28. The aptamerof claim 17 consisting of RNA.
 29. The aptamer of claim 17 consisting ofDNA.
 30. The aptamer of claim 17 wherein X represents a sequenceselected from TGT, GCA and TGA.
 31. The aptamer of claim 17 wherein D₁and D₁N are selected from the following respective pairs: CAG and CTG;CAGC and GCTG; CATGC and GCATG; CATCGC and GCGATG.
 32. The aptamer ofclaim 17 wherein D₂ and D₂N are selected from the following respectivepairs: CAC and GTG; GCAC and GTGC; GCTAC and GTAGC; GACTAC and GTAGTC.33. Aptamers selected from those with the following sequences: DH6-1 5′p CTG GGT TGG TGA GGT (SEQ ID NO: 4) TGG TCA GCA CGG TTG GTG AGG TTG GTGTG 3′ DH8-1 5′ p GCT GTG GTT GGT GAG (SEQ ID NO: 5) GTT GGC AGC GCA CTGGTT GGT GAG GTT GGG TGC 3′ DH10-1 5′ p GCA TGT GGT TGG TGA (SEQ ID NO:6) GGT TGG CAT GCG CTA CTG GTT GGT GAG GTT GGG TAG C 3′ DH12-1 5′ p GCGATG TGG TTG GTG (SEQ ID NO: 7) AGG TTG GCA TCG CGA CTA CTG GTT GGT GAGGTT GGG TAG TC 3′ TS1-1 5′ p GCT GTG GTT GGT GAG (SEQ ID NO: 8) GTT GGCAGC AGC CAA GGT AAC CAG TAC AAG GTG CTA AAC GTA ATG GCT TCG GCT 3′ TS2-15′ p GCT GTG GTT GGT GAG (SEQ ID NO: 17) GTT GGC AGC AGC TGG CGG TAC GGGCCG TGC ACC CAC TTA CCT GGG AAG TGA GCT 3′ TS3-1 5′ p GCT GTG GTT GGTGAG (SEQ ID NO: 18) GTT GGC AGC AGC CAT TCA CCA TGG CCC CTT CCT ACG TATGTT CTG CGG GTG GCT 3′ DH8-Br 5′ GCT GTG GTT GGB GAG (SEQ ID NO: 12) GBBGGC AGC GCA CBG GBB - GGB GAG GBB GGG BGC 3′

where B=5-bromo-2′-deoxyuridine, 5-iodo-2′-deoxyuridine or otherphotoactive nucleotide analogue
 34. An antidote aptamer comprising atleast ten nucleotides and/or nucleotide analogues complimentary to asequence of at least ten nucleotides and/or nucleotide analogues from anaptamer according to claim
 17. 35. An antisense oligonucleotide to anaptamer of claim
 17. 36. An antidote aptamer according to claim 34ligated at its termini to form a circular oligonucleotide.
 37. Theantidote aptamer or the antisense oligonucleotide of claim 36 whereinthe termini have been chemically ligated.
 38. The antidote aptamer orthe antisense oligonucleotide of claim 37 wherein the termini have beenenzymatically ligated.
 39. An aptamer according to claim 34 having thefollowing sequence: ADH8-1 5′ pGCA CCC AAC CTC ACC AAC (SEQ ID NO: 19)CAG TGC GCT GCC AAC CTC ACC AAG CAC AGC 3′.


40. A method of treatment of thrombosis in a patient requiring suchtreatment which comprises administering to said patient an effectiveamount of an aptamer according to claim
 1. 41. A method of preventing orreducing coagulation of blood or blood derived products which comprisescontacting the blood or blood derived product with an effective amountof an aptamer according to claim
 1. 42. Use of a compound according toclaim 1 in preparation of a medicament for the treatment of thrombosis.43. A method for capturing leukocytes from a physiological fluidcomprising contacting the physiological fluid with an effective amountof an aptamer according to claim
 1. 44. A composition comprising anaptamer according to claim 1 or its antisense antidote together with oneor more pharmaceutically acceptable carriers or excipients.
 45. Acomposition according to claim 41 in oral dosage form.
 46. A method forcounteracting the effect of an aptamer according to claim 1 comprisingcontacting the aptamer with a counteracting effective amount of anantidote aptamer thereof.
 47. An antisense oligonucleotide according toclaim 35 ligated at its termini to form a circular oligonucleotide.